Juliette Paillet, Alexandre Duprey, Emiel van der Kouwe
This gene sequence includes both of EpCAM Binding Docks; BBa_K772001 and BBa_K772002. Thus, this composite part is the main component of CTC (Circulating Tumor Cell) detection system. Docks include an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C or N terminus. Hopefully, after binding to the EpCAM antigen, TEV protease domains will fix together to form the full structure in order to activate the signalling pathway. (For more information: BBa_K772003) EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequences, there are a constitutive pLac promoter and appropriate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the domain of TEV protease.
CD225 (EpCAM antibody) is commonly used for the test of presence of EpCAM in the laboratories. The sequences of OmpA and TEV protease terminuses are acquired from the Registry.
CBD of C. cellulovorans cellulose binding protein gene with const. Promoter (J23100+J61101) and GFP
Cellulose binding Domain of Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)] with a constitutive promoter and RBS ( J61101 ) and a fused-GFP protein with His-tag.
Was made to switch from a T7-promoter to a constitutive promoter
Cellulose binding domain form a cloning-vector. J61101 and [http://partsregistry.org/Part:BBa_I13522 GFP] from the partsregistry.
P24 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
Constructs were assembled according to R.F.C. 25. All parts were codon optimized for E. coli.
This gene sequence includes an EpCAM (Epithelial p? Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease N terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease C terminus differently instead of N terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the part of TEV protease.
P13 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from Escherichia coli. The protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. The protein to be a dimer in the absence of CO. The product, CooA, is 28% identical (51% similar) to CRP(cAMP receptor protein) and 18% identical (45% similar) to FNR(fumavate nitrate reduction) from Escherichia coli. Inactive Fe(II) CooA structure adapted from that of the strain with PDB identification no. 1FT9. The protein consists of two monomers, shaded differently, which dimerize along the central C-helices of adjacent effector-binding domains. The solved structure is asymmetric, in which one monomer contains fused C- and D-helices. Nonetheless, both F-helices that interact with DNA in a sequence-specific manner are buried from the surface in the structure. The 4/5 loop is noted and so are the Pro2 and His77 heme Fe(II) ligands.
The sequence information was acquired from NCBI and physical DNA was synthesized from GENEART. Classical cloning strategies(restriction digestion) were used to produce biobricks.
Hwan Youn, Robert L. Kerby, Mary Conrad, et al. 2004. Functionally Critical Elements of CooA-Related CO Sensors. J. Bacteriol. 186(5):1320-1329. doi:10.1128/JB.186.5.1320-1329.2004.
Amino acids 2-64 from the zebrafish CBX2 protein. This is the conserved Polycomb chromodomain predicted to bind trimethylated histone H3 lysine 27 in chromatin.
Codons were optimized for expression in cultured human cells.
Danio rerio (zebrafish) CBX2 amino acids 2-64. The human codon-optimized coding sequence was synthesized by Genscript.
P21 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
P15 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
p70-CsgA-His*2, double His-tagged curli generator
P05 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
P04 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
GAL4 based yeast light-switchable promoter system
BBa_K801040 (SV40NLS-PhyB-Linker-GAL4DBD)
20 amino acids long linker (BBa_K365005)
BBa_K801039 (SV40NLS-GAL4AD-Linker-PIF3)
SV40 NLS (SV40 nuclear localization signal)
Phytochrome B (PhyB, 908 first N-terminal amino acids)
GAL4AD (GAL4 transcriptonal activation domain)
Modified FKBP12-rapamycin-binding domain (FRB) of human mTOR Kinase (=mammalian Target of Rapamycin, also called FRAP = FKBP12-rapamycin associated protein). In presence of the immunosupressant drug rapamycin, FRB forms a high-affinity complex with FKBP12. There is no detectable FKBP12 - FRB interaction in absence of rapamycin. ===Modification for a non-toxic dimerizer=== Rapamycin arrests growth and proliferation of many cell types (reviewed in Jacinto and Hall, 2003) by inhibiting protein translation. The mutation T2089L (mTor numbering) makes FRB binding to a non-toxic Rapamycin analogue (rapalogue) AP21967 as well as the original Rapamycin. The FRB - FKBP12 heterodimerization can then be triggered by both Rapamycin and the non-toxic rapalogue. This variant of FRB and the synthetic dimerizer AP21967 are the basis of the Argent(tm) heterodimerization kit from Ariad Pharmaceuticals Inc. '''Note:''' Two additional mutations would convert FRB into FRB* which is used for conditional (drug-reversible) degradation of fusion proteins. These two additional mutations are not implemented in this part. ===Protein Parameters:=== * Number of amino acids: 93 * Molecular weight: 11285.9 * Theoretical pI: 6.47
* aa translation from human mTOR, * mutation T2089L (verified against pC4-RHE from Ariad) * GeneArt codon optimization for E. coli * gene synthesis
human mTOR (aa sequence) gene synthesis
P23 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
SV40NLS-GAL4AD-linker-PIF3 (part for GAL4/LexA based light-switchable promoter system)
CDS for a synthetic hybrid fusion protein. Consists of serveral domains. RFC10 pre and suffix
CDS for a synthetic hybrid fusion protein
SV40 nuclear localization sequence (recognized by importin alpha)
Cellulose binding Domain of Cellulomonas Fimi Exoglucanse (Freiburg-Standard)
Cellulose binding domain of the Cellulomonas fimi ATCC 484 exoglucanase gene
The sequence starts with the Freiburg-prefix (ATG+NgoMIV-restrictionsite) followed by 12 Bases (4 AS) of the exoglucanase gene in front of the CBD. Then 9 bases of the exoglucanase gene behind the CBD. After that the sequence continues with a short GS-Linker.
Cloning-Vector with a CBDcex-Barnase fusion-protein
P20 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
The immunophilin protein FKBP12 (FK506-binding protein) is the target of the natural compounds FK506 and rapamycin. The FKBP/rapamycin or FKBP/FK506 complex then binds to several other proteins and suppresses immune reactions. Drug screening and design has turned up a selection of other small molecules that bind with high specificity to FKBP12 itself or to mutants of FKBP12. A second human protein, FRB is among the most prominent binding targets of the FKBP/rapamycin complex (using the "other face" of the rapamycin molecule). Rapamycin can herefore induce the hetero-dimerization of FKBP12 and FRB. == See also == * structure of FRB / FKBP / Rapamycin complex: PDB 1nsg (CRG-internal ID: rg0073)
* gene synthesis * codon optimized for E. coli
PoPS --> N4 gp8 (DNA replication inhibitor)
Rashmi Sharma, Bryce Bajar, Kendrick Wang
Fuse lambda cI with LGF2 mutated from Glu to Ala at site 75 (the Glu which locates after three continuous Ala behind lambda repressor is at site 68). As one of the components in Bacterial Two-hybrid System, it causes little interaction with GAL11P compared to the normalized WT.
The LGF2 is mutated from GAA to GCA at site 75.
It is from Agilent Technologies' BacterioMatch ⅡTwo-Hybrid System Vector Kit and modified by Site-Directed Mutagenesis(SDM).
Part consists of the signal peptide of Erbb1/EGF-receptor, which ensures the transport to the cell membrane scFV-antiNIP, GGGSflexlinker (extracellular parts), the trans-membrane region of the Erbb1/EGF-receptor and the N-terminal part of the split-fluorophor YFP(intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157008. For further information see the description of the parts mentioned above.
A composite part consisting of the signal-peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the N-terminal part of split-enzyme beta-Lactamase (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and BBa_I757011. For further information see the description of the parts mentioned above.
N-terminal fragment of H. gaussia luciferase for PCA. '''Disulfide bonds:''' * likely, many Cys in sequence ===Purification=== Inouye & Sahara (2008) discuss the purification and in-vitro activity of different Luciferases, including Gaussia. They indicate that Gaussia Luciferase was previously purified from insoluable fractions -- perhaps due to the high Cys content. Goerke et al (2008) Provide optimized in-vitro expression conditions for Gaussia Luciferase based on tuning the strain from which the cell-free lysate is produced. ===References=== http://www.ncbi.nlm.nih.gov/pubmed/17099704 Remy I, Michnick SW. (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase. http://nar.oxfordjournals.org/cgi/content/full/27/13/e4#hd1 Maroun M, Aronheim A. (2007) A novel in vivo assay for the analysis of protein-protein interaction. PMID: 10373602 http://www.ncbi.nlm.nih.gov/pubmed/19373229 Tannous BA. (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. http://www.ncbi.nlm.nih.gov/pubmed/18789309 Inouye S, Sahara Y. (2008) Soluble protein expression in E. coli cells using IgG-binding domain of protein A as a solubilizing partner in the cold induced system. http://www.ncbi.nlm.nih.gov/pubmed/18555198 Goerke AR, Loening AM, Gambhir SS, Swartz JR. (2008) Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase.
Part consists of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split fluorophor YFP with a fluolinker joined to its N-Terminus (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and Part 2 of Split- Luciferase For further information see the description of the parts mentioned above.
Fluoresceine A -binding derivative of a Lipocalin (BBP, bilin binding protein from pieris brassicae), originally designed by Arne Skerra [see Ref. 1,2].
Between BioBrick 1.0 sites this part is flanked 5' by NgoMI(=NgoMIV) and 3' AgeI(=PinAI) to facilitate in frame cloning for protein fusions.
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
P16 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
P11 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
P22 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
Cellulose binding domain of C. cellulovorans cellulose binding protein gene (Freiburg-Standard)
Cellulose binding Domain of Clostridium cellulovorans cellulose binding protein gene (cbp A) http://www.ncbi.nlm.nih.gov/nuccore/M73817
The sequence starts with the Freiburg-prefix (ATG+NgoMIV-restriction-site) followed by 12 Bases (4 AS) of the cellulose binding protein gene in front of the CBD; then 12 bases (4 amino acids) of the cellulose binding protein gene behind the CBD; after that the sequence continues with a short GS-Linker and ends with the Freiburg-suffix (AgeI-restirction-site+TAA).
Coding region for cellulose binding domain. When fused to a protein, protein attaches to cellulose
Sequence was optimized for Escherichia coli and Arabidopsis thaliana codon usage.
Domain derived from cellulose binding protein from Chlostridium cellulovorans(WP_013291799).
Engineered Leu zipper based on VBP B-ZIP domain that forms medium-weak parallel heterodimers with variants E34(X). This zipper corresponds to RR34(A) in Acharya et al and R34 in Bashor et al. Bashor et al. used the three different pairs to tune the affinity of their MAPK feedback device. Interaction with E34(X) variants: * E34(I): Kd = 6.1 nM = J18923 * E34(V): Kd = 41 nM * E34(N): Kd = 810 nM == References == * Acharya et al. (2002) A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I, V, L, N, A, and K. PMID: 12450375 * Bashor et al (2008) Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics. PMID: 18339942
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and Part one of Split- Luciferase For further information see the description of the parts mentioned above.
P01 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
This gene sequence includes an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease N terminus differently instead of C terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
The sequence codes for cellulose binding domain cbh2 in prokaryotic cells.
Restriction sites Xho I and Bamh I are added.
Fuse lambda cI with LGF2 mutated from Asp to Ala at site 78. As one of the components in Bacterial Two-hybrid System, it causes a less interaction with GAL11P compared to the nomalized WT.
From Agilent Technologies' BacterioMatch ⅡTwo-Hybrid System Vector Kit.
E34(I) strong heterodimerizing leucine zipper
Engineered Leu zipper based on VBP B-ZIP domain that forms strong parallel heterodimers with R34. Interaction with R34: * Kd = 6.1 nM References ============ * Acharya et al. (2002) A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I, V, L, N, A, and K. PMID: 12450375 * Bashor et al (2008) Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics. PMID: 18339942
SV40NLS-PhyB-linker-Gal4DBD (part for GAL4 based light switchable promoter system)
Composite part of BBa_K801030, BBa_K801030, BBa_K365005, BBa_K801035
Essential Cys for chromophore attachement of phycocyanobilin (PCB)
Essential Gly for photoconvertible binding of PIF3
Essential Arg for photoconvertible binding of PIF3
Cellulose binding Domain of C. cellulovorans cellulose binding protein with Reporter GFP
Cellulose binding Domain from Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene] with T7, RBS linked with two amino acids (GS) to a GFP with His-tag.
CBDclos fused to a his-tagged GFP in a Freiburg-manner
Cloning-vector and [http://partsregistry.org/Part:BBa_K863121 BBa_K863121]
composite part of Bba_K365005 and Bba_K365000
Phytochrome B (PhyB, first 908 N-terminal amino acids)
improved version of BBa_K365002 to allow N- and C-terminal protein fusions using RFC 25
Essential Cys for chromophore attachement of phycocyanobilin/phytochromobilin
Essential Arg for photoconvertible binding of PIF3/6
Essential Gly for photoconvertible binding of PIF3/6
Fuse lambda cI with LGF2 mutated from Arg to Ala at site 74 (the Glu which locates after three continuous Ala behind lambda repressor is at site 58). As one of the components in Bacterial Two-hybrid System, it causes a stronger interaction with GAL11P compared to the normalized WT.
The LGF2 is mutated from CGA to GCA at site 74.
Composite part of BBa_K801031, Bba_K365005 and BBa_K801032
Cellulose binding Domain from C. Fimi with const. Promotor and GFP
Cellulose binding Domain from Cellulomonas Fimi with const. Promotor J61101 and Reporter GFP
Fuse lambda cI with LGF2 mutated from Asp to Ala at sites 76 and 78 (the Glu which locates after three continuous Ala behind lambda repressor is at site 58). As one of the components in Bacterial Two-hybrid System, it causes little interaction with GAL11P compared to the normalized WT.
The LGF2 is mutated from GAC to GCC at site 76 and from GAC to GCC at site 78.
Top metal binding protein. Binds zinc, arsenic, copper and cadmium.
Multiple domain protein. Domains bind zinc, arsenic, copper and cadmium. Sequences derived from metallothioneins and transcription factors of different organisms. Arsenic, zinc and cadmium binding regions are full length metallothioneins. Zinc binding region derived from a transcritpion factor binding domain. Different protein binding regions are separated with glycerin-serine spacers to improve domain folding. Metal binding regions consists of disulfide bonds. For expression in E. coli disulfide bond forming strains are useful (e. g. Origami).
Arsenic (CAA06729), zinc (CAD88267) and cadmium (AAN06822) metallothioneins are full length. Copper AAA34542)domain is partial.
P18 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
P25 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
The second LOV domain of Arabidopsis phototropin 1 shows light-induced transient (30s) homo-dimerization between a ground state and an excited monomer (Nakasone et al. 2006). LOV2 is predominantly monomeric at concentrations below 100microM but turns dimeric above. Funnily, the high-conc. dimer state experiences photo-dissociation instead. Either the excitation follows a different course from the dimer starting state or the excitation event itself pushes the complex apart. (CRG-internal ID: rg0090) ===References=== * Nakasone et al. 2006 PMID: 16679373 * Freddolino, Peter L.; Dittrich, Markus; Schulten, Klaus (2006) Dynamic Switching Mechanisms in LOV1 and LOV2 Domains of Plant Phototropins PMID: 16935961
The fragment used by Nakasone et al. is 475K - 578G. The core domain 449E - 586R has reportedly lower stability at RT (aggregation). * gene synthesis * codon optimized for E. coli
P19 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
* Parts were assembled according to R.F.C. 25. * All parts were codon optimized for E. coli
Cellulose binding Domain from Clostridium cellulovorans cellulose binding protein
Cellulose binding Domain from Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene] in Freiburg-Standard.
Added Freiburg-pre- and suffix for in frame assembly and a two amino acids (GS)-Linker.
Singlechain Fv.Fragment of an Anti-Nitro-Iodo-Phenol-antibody B1-8. Binds the hapten NIP (Nitro-Iodo-Phenole) with an affinity of 2.0 uM[1,2]. Designed for fusion to proteins or peptides via in frame cloning.
P08 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
DNA Polymerase Inhibitor (via N4 coliphage gp8)
P09 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
NOD1 (ncbi: https://www.ncbi.nlm.nih.gov/gene/10392)
Cellulose binding domain of C. cellulovorans with T7, RBS, GS-Linker (Freiburg-Standard)
Cellulose binding domain of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N Cellulomonas fimi ATCC 484 exoglucanase gene] in Freiburg-standard
Added the regulatory unit BBa_K525998 (T7+RBS) in front of the CBD and a short GS-Linker behind the domain and used Freiburg-Standard to be able to fuse proteins to the domain.
Cellulose binding Domain of C. Fimi Exoglucanase with T7, RBS, GS-Linker (Freiburg-Standard)
Cellulose binding domain of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N Cellulomonas fimi ATCC 484 exoglucanase gene] in Freiburg-standard with T7, RBS, GS-Linker (Freiburg-Standard)
You can find additional info to the Cellulose binding domain here: [http://partsregistry.org/Part:BBa_K863101:Design CBDcex_Freiburg]
Human Polycomb chromodomain from the CBX8 protein (a.a. 1-63). See Haynes and Silver, JBC, 2011
P12 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
P10 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
P03 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157009 and BBa_K157005. For further information see the description of the parts mentioned above.
composite part of Bba_K365000 and Bba_K365005
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157006. For further information see the description of the parts mentioned above.
A composite part consisting of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split-enzyme beta-Lactamase (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and BBa_I757012. For further information see the description of the parts mentioned above.
P02 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
Constructs were assembled according to R.F.C. 25 All parts were codon optimized for E. coli
SV40NLS-PhyB-linker-LexA (part for a LexA based light-switchable promoter system)
Composite part of BBa_K801030, BBa_K801030, BBa_K365005, BBa_K801033
P17 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
RNA binding protein, recognises and binds RNA sequence:
p70-CsgA-His, His-tagged curli generator
C-terminal fragment of H. gaussia luciferase for PCA (detection of protein-protein interactions). See part J18928 for details.
P06 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices
LexA based yeast light-swithable promoter system
BBa_K365005 (20 amino acids long linker)
Cellulose binding Domain from Cellulomonas Fimi with Reporter GFP
Cellulose binding Domain of C. Fimi Exoglucanase gene with T7, RBS linked with two amino acids (GS) to a GFP (Freiburg-Standard)
CBDcex fused to a his-tagged GFP in a Freiburg-manner
A composite part consisting of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split fluorophor YFP with a fluolinker joined to its N-Terminus (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157009 and BBa_K157007. For further information see the description of the parts mentioned above.
The scFv-anti-NIP (with a flexible GGGS-linker at the C-terminus) fused to the signal peptide of Erbb1/EGF-receptor.
Derived by cloning of the parts BBa_K157001, BBa_K157003 and BBa_K157013. For further information see the description of the parts mentioned above.